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Identification of motifs that function in the splicing of non-canonical introns.

Murray JI, Voelker RB, Henscheid KL, Warf MB, Berglund JA - Genome Biol. (2008)

Bottom Line: While the current model of pre-mRNA splicing is based on the recognition of four canonical intronic motifs (5' splice site, branchpoint sequence, polypyrimidine (PY) tract and 3' splice site), it is becoming increasingly clear that splicing is regulated by both canonical and non-canonical splicing signals located in the RNA sequence of introns and exons that act to recruit the spliceosome and associated splicing factors.In vivo splicing studies show that C-rich and G-rich motifs function as intronic splicing enhancers in a combinatorial manner to compensate for weak PY tracts.The enrichment of specific intronic splicing enhancers upstream of weak PY tracts suggests that a novel mechanism for intron recognition exists, which compensates for a weakened canonical pre-mRNA splicing motif.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Chemistry, Institute of Molecular Biology, University of Oregon, Eugene, Oregon, USA.

ABSTRACT

Background: While the current model of pre-mRNA splicing is based on the recognition of four canonical intronic motifs (5' splice site, branchpoint sequence, polypyrimidine (PY) tract and 3' splice site), it is becoming increasingly clear that splicing is regulated by both canonical and non-canonical splicing signals located in the RNA sequence of introns and exons that act to recruit the spliceosome and associated splicing factors. The diversity of human intronic sequences suggests the existence of novel recognition pathways for non-canonical introns. This study addresses the recognition and splicing of human introns that lack a canonical PY tract. The PY tract is a uridine-rich region at the 3' end of introns that acts as a binding site for U2AF65, a key factor in splicing machinery recruitment.

Results: Human introns were classified computationally into low- and high-scoring PY tracts by scoring the likely U2AF65 binding site strength. Biochemical studies confirmed that low-scoring PY tracts are weak U2AF65 binding sites while high-scoring PY tracts are strong U2AF65 binding sites. A large population of human introns contains weak PY tracts. Computational analysis revealed many families of motifs, including C-rich and G-rich motifs, that are enriched upstream of weak PY tracts. In vivo splicing studies show that C-rich and G-rich motifs function as intronic splicing enhancers in a combinatorial manner to compensate for weak PY tracts.

Conclusion: The enrichment of specific intronic splicing enhancers upstream of weak PY tracts suggests that a novel mechanism for intron recognition exists, which compensates for a weakened canonical pre-mRNA splicing motif.

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G-rich motifs function as ISEs in LCAT intron 4 splicing. (a) LCAT intron 4 with the mutations shown in blue above the WT sequence. BPS, branchpoint. (b) Splicing of the LCAT intron 4 mini-genes (WT, MUT3, MUT4, MUT7 and MUT 5) in HeLa cells. Splicing products (isolated from HeLa, reverse-transcribed and amplified with radioactive PCR) were resolved on an 8% non-denaturing gel and scanned using a phosphorimager. The pre-mRNA (top) is a 472 bp product and the mRNA (bottom) is a 389 bp product. The average quantification and standard deviation of the percent pre-mRNA (pre-mRNA divided by total RNA) for at least triplicate reactions is reported below each lane. (c) Graphical representation of the percent pre-mRNA for each LCAT mini-gene. Error bars represent standard deviation of replicate experiments.
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Figure 4: G-rich motifs function as ISEs in LCAT intron 4 splicing. (a) LCAT intron 4 with the mutations shown in blue above the WT sequence. BPS, branchpoint. (b) Splicing of the LCAT intron 4 mini-genes (WT, MUT3, MUT4, MUT7 and MUT 5) in HeLa cells. Splicing products (isolated from HeLa, reverse-transcribed and amplified with radioactive PCR) were resolved on an 8% non-denaturing gel and scanned using a phosphorimager. The pre-mRNA (top) is a 472 bp product and the mRNA (bottom) is a 389 bp product. The average quantification and standard deviation of the percent pre-mRNA (pre-mRNA divided by total RNA) for at least triplicate reactions is reported below each lane. (c) Graphical representation of the percent pre-mRNA for each LCAT mini-gene. Error bars represent standard deviation of replicate experiments.

Mentions: We examined the role of two G-rich motifs (G-rich motif (GRM)1 and GRM2) present upstream of the PY tract of LCAT intron 4 (Figure 4a). The wild-type (WT) LCAT intron 4 mini-gene splices such that 5 ± 1% pre-mRNA is observed (Figure 4b, lane 1, and 4c). Mutation of GRM1 to AAA (MUT 3, Figure 4a) had a strong effect, and increased the unspliced product to 19 ± 5% (Figure 4b, lane 2, and 4c). Mutation of GRM2 to AAA (MUT 4, Figure 4a) had slightly less of an effect than MUT 3, resulting in 14 ± 3% pre-mRNA (Figure 4b, lane 3, and 4c). Mutation of both GRM1 and GRM2 (MUT 7, Figure 4a) had a similar effect as mutation of GRM1 alone (Figure 4b, lane 4, and 4c), suggesting that the two GRMs do not function additively towards recognition of LCAT intron 4. We also mutated a region that was neither a G-rich motif nor C-rich motif (MUT 5, Figure 4a) to be sure that the AAA motif we were inserting was not acting as an ISS. MUT 5 spliced similarly to WT (Figure 4b, compare lanes 1 and 5; Figure 4c), suggesting that the presence of the mutant AAA sequence in that region of LCAT intron 4 does not act as an ISS. These results suggest that GRM1 and GRM2 are ISEs important for the splicing of LCAT intron 4.


Identification of motifs that function in the splicing of non-canonical introns.

Murray JI, Voelker RB, Henscheid KL, Warf MB, Berglund JA - Genome Biol. (2008)

G-rich motifs function as ISEs in LCAT intron 4 splicing. (a) LCAT intron 4 with the mutations shown in blue above the WT sequence. BPS, branchpoint. (b) Splicing of the LCAT intron 4 mini-genes (WT, MUT3, MUT4, MUT7 and MUT 5) in HeLa cells. Splicing products (isolated from HeLa, reverse-transcribed and amplified with radioactive PCR) were resolved on an 8% non-denaturing gel and scanned using a phosphorimager. The pre-mRNA (top) is a 472 bp product and the mRNA (bottom) is a 389 bp product. The average quantification and standard deviation of the percent pre-mRNA (pre-mRNA divided by total RNA) for at least triplicate reactions is reported below each lane. (c) Graphical representation of the percent pre-mRNA for each LCAT mini-gene. Error bars represent standard deviation of replicate experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 4: G-rich motifs function as ISEs in LCAT intron 4 splicing. (a) LCAT intron 4 with the mutations shown in blue above the WT sequence. BPS, branchpoint. (b) Splicing of the LCAT intron 4 mini-genes (WT, MUT3, MUT4, MUT7 and MUT 5) in HeLa cells. Splicing products (isolated from HeLa, reverse-transcribed and amplified with radioactive PCR) were resolved on an 8% non-denaturing gel and scanned using a phosphorimager. The pre-mRNA (top) is a 472 bp product and the mRNA (bottom) is a 389 bp product. The average quantification and standard deviation of the percent pre-mRNA (pre-mRNA divided by total RNA) for at least triplicate reactions is reported below each lane. (c) Graphical representation of the percent pre-mRNA for each LCAT mini-gene. Error bars represent standard deviation of replicate experiments.
Mentions: We examined the role of two G-rich motifs (G-rich motif (GRM)1 and GRM2) present upstream of the PY tract of LCAT intron 4 (Figure 4a). The wild-type (WT) LCAT intron 4 mini-gene splices such that 5 ± 1% pre-mRNA is observed (Figure 4b, lane 1, and 4c). Mutation of GRM1 to AAA (MUT 3, Figure 4a) had a strong effect, and increased the unspliced product to 19 ± 5% (Figure 4b, lane 2, and 4c). Mutation of GRM2 to AAA (MUT 4, Figure 4a) had slightly less of an effect than MUT 3, resulting in 14 ± 3% pre-mRNA (Figure 4b, lane 3, and 4c). Mutation of both GRM1 and GRM2 (MUT 7, Figure 4a) had a similar effect as mutation of GRM1 alone (Figure 4b, lane 4, and 4c), suggesting that the two GRMs do not function additively towards recognition of LCAT intron 4. We also mutated a region that was neither a G-rich motif nor C-rich motif (MUT 5, Figure 4a) to be sure that the AAA motif we were inserting was not acting as an ISS. MUT 5 spliced similarly to WT (Figure 4b, compare lanes 1 and 5; Figure 4c), suggesting that the presence of the mutant AAA sequence in that region of LCAT intron 4 does not act as an ISS. These results suggest that GRM1 and GRM2 are ISEs important for the splicing of LCAT intron 4.

Bottom Line: While the current model of pre-mRNA splicing is based on the recognition of four canonical intronic motifs (5' splice site, branchpoint sequence, polypyrimidine (PY) tract and 3' splice site), it is becoming increasingly clear that splicing is regulated by both canonical and non-canonical splicing signals located in the RNA sequence of introns and exons that act to recruit the spliceosome and associated splicing factors.In vivo splicing studies show that C-rich and G-rich motifs function as intronic splicing enhancers in a combinatorial manner to compensate for weak PY tracts.The enrichment of specific intronic splicing enhancers upstream of weak PY tracts suggests that a novel mechanism for intron recognition exists, which compensates for a weakened canonical pre-mRNA splicing motif.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Chemistry, Institute of Molecular Biology, University of Oregon, Eugene, Oregon, USA.

ABSTRACT

Background: While the current model of pre-mRNA splicing is based on the recognition of four canonical intronic motifs (5' splice site, branchpoint sequence, polypyrimidine (PY) tract and 3' splice site), it is becoming increasingly clear that splicing is regulated by both canonical and non-canonical splicing signals located in the RNA sequence of introns and exons that act to recruit the spliceosome and associated splicing factors. The diversity of human intronic sequences suggests the existence of novel recognition pathways for non-canonical introns. This study addresses the recognition and splicing of human introns that lack a canonical PY tract. The PY tract is a uridine-rich region at the 3' end of introns that acts as a binding site for U2AF65, a key factor in splicing machinery recruitment.

Results: Human introns were classified computationally into low- and high-scoring PY tracts by scoring the likely U2AF65 binding site strength. Biochemical studies confirmed that low-scoring PY tracts are weak U2AF65 binding sites while high-scoring PY tracts are strong U2AF65 binding sites. A large population of human introns contains weak PY tracts. Computational analysis revealed many families of motifs, including C-rich and G-rich motifs, that are enriched upstream of weak PY tracts. In vivo splicing studies show that C-rich and G-rich motifs function as intronic splicing enhancers in a combinatorial manner to compensate for weak PY tracts.

Conclusion: The enrichment of specific intronic splicing enhancers upstream of weak PY tracts suggests that a novel mechanism for intron recognition exists, which compensates for a weakened canonical pre-mRNA splicing motif.

Show MeSH
Related in: MedlinePlus